It has been proposed that transition of cancer cells to a metastatic phenotype within the primary tumor site involves the loss of the epithelial phenotype and the acquisition of a mesenchymal (fibroblast-like) phenotype (epithelial-mesenchymal transition: EMT). A crucial role for the tumor microenvironment (hypoxia, stromal factors, inflammation) in the induction of EMT has been proposed. Thus, adaptive rather than selective pressures are operative. Based on the retarded growth of EMT converted cells, epithelial function needs to be regained at the metastatic site to facilitate tumor growth. This has led to the assumption that EMT is reversible and mesenchymal-epithelial transition (MET) occurs at the metastatic site. These experimental observations are consistent with pathologic examination of human cancer tissues revealing remarkable resemblance between primary and metastatic tumors. Experimental models and clinical correlations of different types of cancers have implicated numerous mechanistic factors in EMT;these data suggest that a "one size fits all" approach is overly simplistic. Membrane type 1-matrix metalloproteinase (MT1-MMP) is among the most prominent factors implicated in cancer invasion and metastasis. Based on data from several labs including our own, we propose that microenvironmental factors within primary prostate cancers, result in activation of MT1-MMP on the cancer cell surface, which initiates a series of events, leading to EMT and metastasis. The signaling pathways involved in MT1-MMP induced EMT include Wnt5a, Rac1, and generation of reactive oxygen species (ROS). We propose that following removal of the initiating events e.g. hypoxia, downregulation of MT1-MMP occurs at the metastatic site, leading to MET. The primary goal of this proposal is to characterize the MT1-MMP, Wnt5a, ROS pathway leading to EMT/MET and metastasis of prostate cancer. To accomplish this goal, we will employ our recently published prostate cancer model displaying MT1-MMP induced EMT. Expression of MT1-MMP in human LNCaP prostate cancer cells induces EMT-like phenotypic changes associated with loss of E-cadherin at the primary tumor site. Using cDNA microarray and shRNA strategies, we demonstrated that Wnt5a is a downstream effector of MT1-MMP induced EMT. Reactive oxygen species are increased in this EMT model, leadig to DNA damage. The three major aims proposed in this grant are to: (1) Characterize the reversibility and mechanism of MT1-MMP/Wnt5a Induced EMT in Prostate Cancer;(2) Delineate the role of hypoxia and the generation of ROS in the EMT Pathway of Prostate Cancer. Assess the role of mitochondrial versus NAD(P)H oxidase sources of ROS in prostate cancer. Examine the role of ROS in inducing genomic instability. (3) Employ immunohistochemial markers of EMT and hypoxia in human prostate cancer specimens and determine if these markers correlate with stage of disease and biochemical failure (rise in PSA). PUBLIC HEALTH RELEVANCE: Prostate cancer is the major visceral (non skin) cancer diagnosis (20% of all cancers) and a leading cause of morbidity and mortality among men. It is the second leading cause of cancer mortality among American men. Prostate cancer is especially prominent among elderly veterans, with more than 11,000 new cases diagnosed per year in VA hospitals (VA Central Office data). Although surgery and radiation therapy are effective in early stage prostate cancer, treatment options are limited for patients with metastatic disease who become unresponsive to hormonal (androgen deprivation) therapy. The recent introduction of Taxotere chemotherapy for metastatic prostate cancer only prolongs survival by 2 to 3 months. In order to develop new approaches to the treatment of advanced prostate cancer, it is essential that scientists better understand basic mechanisms leading cancers to spread from the prostate gland to other parts of the body (metastasis). Over the past few years, it has been recognized that cancer cells in the prostate gland begin to change their shape and become more aggressive over time. This change is called, "epithelial-to- mesenchymal transition", abbreviated as EMT. It is widely recognized that EMT may represent an important mechanism leading to metastasis. Hence, it is critical that we understand the details of how molecules working inside cancer cells produce EMT and metastasis. Once we understand how these molecules lead cancer cells to spread around the body, the pharmaceutical industry will be able to develop drugs to counteract these molecules and stop cancer metastasis. This approach will open up new avenues for the control of metastatic prostate cancer. The goal of this grant application is to identify the molecules involved in cancer EMT and to show that interfering with these molecules will stop cancer cells from being able to invade surrounding normal tissues and metastasize around the body. We have recently published an article demonstrating the discovery of a new pathway by which prostate cancer cells become more invasive and capable of metastasis. The current grant will work on demonstrating the detailed molecules involved in prostate cancer EMT and begin to show that experimental drugs can interfere with this process of cancer dissemination. If successful, our research will encourage pharmaceutical companies to develop new drugs based on the experimental approaches that we hope to develop during this grant. These new drugs will be very beneficial to veterans with advanced prostate cancer. This research project will also help to determine which of these "cancer molecules" are increased in prostate cancer biopsy specimens from patients who will or will not develop disseminated cancer in the future. Based on identifying dangerous "cancer molecules" in a patient prostate biopsy specimen, clinical doctors may be able to say which patients need aggressive forms of treatment to prevent metastasis or in the absence of "cancer molecules", which patients can be managed conservatively by watchful waiting rather than surgery (radical prostatectomy) or radiation therapy. The outcome of this research will therefore be very relevance to veterans health and the mission of the VA.